Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing alpha or beta amino acid or substituted amino acid...
Reexamination Certificate
2002-10-02
2004-12-14
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing alpha or beta amino acid or substituted amino acid...
C435S228000, C435S227000, C435S253300, C435S253300, C435S254300, C435S320100, C536S023200, C536S023700
Reexamination Certificate
active
06830905
ABSTRACT:
TECHNICAL FIELD
The present invention relates a novel glutaminase and a gene encoding the same. The glutaminase of the present invention can be utilized as an enzyme for food processing to convert glutamine into glutamic acid exhibiting stronger “umami” taste (umami).
BACKGROUND ART
For the production of soy sauce, miso, and other natural seasonings containing protein hydrolysate products, koji mould (filamentous fungus belonging to the genus
Aspergillus
) has been utilized. For example, soy sauce is produced through two process steps of koji-making and fermentation. In the koji-making step, the starting material is principally degraded by enzymes produced by koji mould. In such a process, it is important to increase the amount of glutamic acid among various tasteful materials in order to obtain stronger umami of soy sauce.
Glutamic acid is produced through two kinds of pathways. The first is the liberation of glutamic acid from protein caused by protease and peptidase. The second is generation of glutamic acid through hydrolysis of glutamine catalyzed by glutaminase (glutamine amidohydrolase).
In the production of soy sauce, liberation ratio of glutamic acid relative to its content in the starting material is not so high, and this is considered to be due to insufficient glutaminase activity of koji mould. Therefore, breeding of strains exhibiting high activities of protease and glutaminase through cell fusion of high protease activity strain and high glutaminase activity strain in solid koji has also been attempted (Ushijima, S. et al., Agric. Biol. Chem., 51 (4), 1051 (1987), Japanese Patent Publication (KOKOKU) No. Hei 3-73271/1992).
As for glutaminase, those derived from various bacteria and animals have been well investigated (Wakayama, M. et al., J. Ferment. Bioeng., 82, No.6, 592-597 (1996), Chung-Bok, Mi, et al., Biochem. J., 324, 193-200 (1997), Duran, S. et al., Biochem. Genet., 34, 453-465 (1996)). On the other hand, investigation about glutaminase of koji mould had been retarded, but extracellular glutaminase and intracellular glutaminase have been purified from one strain of
Aspergillus oryzae
, and they have been characterized (Yano, T. et al., J. Ferment. Technol., Vol. 66, No. 2, 137-143 (1988)). These glutaminases have a molecular weight of about 113,000, and substantially similar properties.
Further, there have been determined an amino acid sequence of N-terminal region of glutaminase derived from
Aspergillus oryzae
HG strain (Fukuoka Industrial Technology Center, Institute of Biology and Food, Research Summary of 1996 (199)), and amino acid sequence within N-terminal region of glutaminase derived from
Aspergillus oryzae
(Food Research Institute, Aichi Prefectural Government, Japan, Annual Report of 1995 (Research Report) pp.3-4, (1996)) for purified glutaminases.
Meanwhile, because koji mould is excellent in the ability for secreting extracellular proteins, it has been attracted attention as a host for the production of recombinant proteins, and practically used for some enzymes.
DISCLOSURE OF THE INVENTION
As described above, koji mould has already afforded results as a material for genetic recombination technology, and its glutaminase has also been investigated to some extent. However, it cannot be considered to be fully investigated, and its further investigation has been desired. In addition, any genes encoding glutaminase of koji mould have not been isolated.
The present invention has been accomplished in view of the aforementioned state of the art, and its object is to provide a gene encoding glutaminase derived from koji mould.
The present inventors successfully purified glutaminase from
Aspergillus oryzae
, determined its partial amino acid sequence, and isolated DNA coding for the glutaminase based on the obtained information, and thus the present invention has been completed. Further, they also succeeded in isolating DNA encoding glutaminase of
Aspergillus nidulans.
That is, the present invention provides the followings:
(1) a protein defined in any of the following (A) to (D):
(A) a protein having an amino acid sequence represented by the amino acid numbers 1-670 of SEQ ID NO: 2 in Sequence Listing;
(B) a protein having an amino acid sequence represented by the amino acid numbers 1-669 of SEQ ID NO: 22 in Sequence Listing;
(C) a protein having an amino acid sequence represented by the amino acid numbers 1-670 of SEQ ID NO: 2 in Sequence Listing with substitution, deletion, insertion, addition or inversion of one or a plurality of amino acids, and having activity for catalyzing hydrolysis of glutamine to glutamic acid and ammonia;
(D) a protein having an amino acid sequence represented by the amino acid numbers 1-669 of SEQ ID NO: 22 in Sequence Listing with substitution, deletion, insertion, addition or inversion of one or a plurality of amino acids, and having activity for catalyzing hydrolysis of glutamine to glutamic acid and ammonia;
(2) a DNA which encodes a protein defined in any of the following (A) to (D):
(A) a protein having an amino acid sequence represented by the amino acid numbers 1-670 of SEQ ID NO: 2 in Sequence Listing;
(B) a protein having an amino acid sequence represented by the amino acid numbers 1-669 of SEQ ID NO: 22 in Sequence Listing;
(C) a protein having an amino acid sequence represented by the amino acid numbers 1-670 of SEQ ID NO: 2 in Sequence Listing with substitution, deletion, insertion, addition or inversion of one or a plurality of amino acids, and having activity for catalyzing hydrolysis of glutamine to glutamic acid and ammonia;
(D) a protein having an amino acid sequence represented by the amino acid numbers 1-669 of SEQ ID NO: 22 in Sequence Listing with substitution, deletion, insertion, addition or inversion of one or a plurality of amino acids, and having activity for catalyzing hydrolysis of glutamine to glutamic acid and ammonia;
(3) the DNA of (2) which is a DNA defined in any of the following (a) to (d):
(a) a DNA which contains nucleotide sequences represented by the nucleotide numbers 1174-1370, 1446-1741, 1800-2242, 2297-2880, 2932-3134, 3181-3324, 3380-3515, 3562-3628 of the nucleotide sequence or SEQ ID NO: 1 in Sequence Listing in this order;
(b) a DNA which contains nucleotide sequences represented by the nucleotide numbers 1807-2000, 2061-2353, 2412-2854, 2915-3498, 3554-3756, 3806-3949, 3996-4131, 4180-4246 of the nucleotide sequence of SEQ ID NO: 21 in Sequence Listing in this order;
(c) a DNA which hybridizes with the DNA of (a) under a stringent condition, and encodes a protein having activity for catalyzing hydrolysis of glutamine to glutamic acid and ammonia;
(d) a DNA which hybridizes with the DNA of (b) under a stringent condition, and encodes a protein having activity for catalyzing hydrolysis of glutamine to glutamic acid and ammonia;
(4) the DNA of (2) which has a nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 17;
(5) the DNA of (3) which has a nucleotide sequence shown in SEQ ID NO: 21 or SEQ ID NO: 25;
(6) a recombinant vector comprising the DNA of (2) inserted in a vector;
(7) a transformant of microorganism introduced with the DNA of (2) in such a manner that the DNA can be expressed to produce glutaminase;
(8) the transformant of (7) which is derived From a filamentous fungus or bacterium belonging to the genus
Escherichia
; and
(9) a method for producing glutaminase which comprises cultivating the transformant of (7) in a culture medium to produce glutaminase in the culture.
The term “glutaminase activity” used in this specification means activity for catalyzing hydrolysis of L-glutamine to L-glutamic acid and ammonia, and the activity may include activity for catalyzing hydrolysis of D-glutamine to D-glutamic acid and ammonia. The activity may also include activities for catalyzing hydrolysis of L-glutamine to L-glutamic acid and ammonia, and D-glutamine into D-glutamic acid and ammonia, and activity for catalyzing transfer reaction or hydrolysis reaction of glutamyl group of L-&ggr;-glutamyl compounds. In the present specification two embodiments are dis
Kataoka Jiro
Kitamoto Katsuhiko
Koibuchi Kyoko
Nagasaki Hiroaki
Yuasa Ari
Ajinomoto Co. Inc.
Nashed Nashaat T.
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